1. Field
This disclosure is concerned generally with fermentation and specifically with the control of dissolved carbon dioxide concentration in mammalian cell fermentation, including factor VIII fermentation, using a culture medium containing complexing agents or mixture thereof with organic buffer.
2. Background
Despite the significant physiological role of dissolved carbon dioxide (DCO2) concentration in mammalian and microbial cell fermentation, this parameter has not been monitored, controlled and optimized efficiently. The DCO2 level in the fermentor affects cell intracellular pH (Andersen et al., 1994), which can potentially disturb a number of metabolic pathways. High DCO2 concentration is a major problem because it can inhibit cell growth (Gray et al., 1996; Kimura et al., 1996), can be detrimental to cell metabolism (Gray et al., 1996) and can decrease product formation (Garnier et al., 1996; Gray et al., 1996; Kimura et al., 1996). In mammalian cells, for example, changes in intracellular pH can influence product quality by altering the glycosylation pattern (Andersen et al., 1994; Kimura et al., 1997). For most cell lines, low DCO2 concentration of about 38 mmHg (5%) is adequate for cell growth (Kilburn, 1991). However, typical DCO2 level in high-density mammalian cell perfusion cultures reaches as high as 180-220 mmHg.
The major problem with DCO2 control in fermentation, especially mammalian cell, is the lack of an appropriate method to keep DCO2 concentration low. Common DCO2 control techniques attempt to remove DCO2 by mechanical means, such as by macrosparging. However, macrosparging is not successful at high cell concentrations. Macrosparging also creates foaming problems and can potentially damage the cells. Although there are some techniques to decrease DCO2, these methods are inadequate, difficult to implement, and inappropriate for scale up to manufacturing level. Previously known techniques, including macrosparging in the fermentor and circulating the broth through external membranes, have the above disadvantages.
Sodium bicarbonate (NaHCO3) is the most common and widely used buffer in cell culture media, but it has inherent disadvantages such as generation of very high DCO2 and suboptimal buffering range due to its low pK value of about 6.3 compared typical cell culture pH range of about 6.8xcx9c7.4. Some mammalian and microbial fermentation media that are free of NaHCO3 have been reported in the literature. See, e.g., Leibovitz, 1963.
The organic buffer MOPS is present in some cell culture media for cultivating mammalian cells, including hybridoma. See the Table. This buffer has also been used in several media used for growing microbial cells, including fungi and bacteria. A few reported media contain a combination of MOPS as buffer at 5-100 mM and a very low concentration of up to about 0.35 mM histidine. Histidine, an amino acid, is present mainly as nutrient supplement in cell culture media and added at a very low concentration of up to about 1.6 mM. Another culture medium contains a combination of iminodiacetic acid (IDA) and very low histidine nutrient supplement. IDA was previously used as a chelating agent for iron in protein-free media for culturing mammalian cells, including hybridoma cells.
We have now discovered a method for the control of DCO2 concentration in mammalian and microbial fermentation. This method comprises culturing the cells in a medium which contains a high concentration of a complexing agent and an organic buffer and which is low in added NaHCO3 concentration. Low NaHCO3 concentration is between 0.01 to 1 g/L.
In a more preferred embodiment the medium contains a high concentration of a complexing agent and an organic buffer and is essentially free of added NaHCO3. Essentially free of added
NaHCO3 means that concentration of added NaHCO3 is less than 0.01 g/L, preferrably 0 g/L. xe2x80x9cAddedxe2x80x9d NaHCO3 means manually added to the medium, as opposed to bicarbonate in the medium as a result of normal cell metabolism. In one preferred embodiment the medium includes 2-40 mM histidine as the complexing agent and 1-40 mM MOPS organic buffer. In another preferred embodiment the medium includes a combination of 1-20 mM iminodiacetic acid complexing agent (in lieu of histidine) and 1-40 mM MOPS organic buffer. In another preferred embodiment the medium includes a combination of 1-20 mM iminodiacetic acid complexing agent and 2-50 mM histidine which acts as the buffer while retaining its complexing property. Both IDA and histidine can have complexing and buffering roles. When both are present in the medium, IDA can act as the primary complexing agent since it is a stronger chelating agent than histidine while histidine can act as the buffer since its pK value of about 6.0 is higher and closer to neutral pH compared with IDA.
Generally, complexing agents form a soluble complex with metal ions. Complexing agents may consist of anionic or neutral molecules. A chelating agent is a subgroup of complexing agents characterized by formation of coordination bonds with metal ions in two (bidentate), three (tridentate) or more positions. For example, histidine is a complexing agent because of the single imidazole ring moiety, while IDA (iminodiacetic acid), EDTA (ethylenediaminetetraacetic acid) and citrate are chelating agents through their multiple carboxylic acid functional groups.
A buffer is a system which resists change in pH when a given increment of H+ or OHxe2x88x92 is added. Buffer systems may consist of organic and/or inorganic buffering species such as MOPS, TES, BES (any Goods buffer) or glucose-6-phosphate as examples of organic buffering systems, and H2PO4xe2x88x92/HPO42xe2x88x92 (phosphate) and H2CO3/HCO3xe2x88x92(bicarbonate) as examples of inorganic buffering systems.
The fermentation pH using the preferred medium is preferably controlled by automatic and in-line addition of an alkali metal hydroxide solution, e.g. 0.1-0.5 M sodium hydroxide, to the medium line. Using the preferred medium and pH control method, metal ions precipitates are prevented from forming during fermentation. This DCO2 control method significantly decreased the DCO2 concentration in the fermentor and maintained the DCO2 at around physiological level even at high cell concentration for a long cultivation period. The preferred medium maintained high cell growth rate and protein production rate when tested on a variety of cell lines, including, e.g., BHK mammalian cells producing recombinant factor VIII (rFVIII).
It is now possible to solve the DCO2 control and optimization problems in a process using a novel cell culture medium which may optionally be used with the above-described pH control method. Details are described below.